Physical and functional probing of DEAD-box proteins as general RNA chaperones

描述（由申请人提供）：从mrna前剪接和翻译到蛋白质和rna的细胞内运输，几乎所有结构化rna进行的过程都需要DExD/H-box蛋白。这些蛋白质被认为利用ATP的能量来促进RNA的构象变化和折叠转变，但在分子水平上对它们如何操纵RNA结构知之甚少。虽然DExD/H-box蛋白的许多RNA和RNA-蛋白靶点大而复杂，难以在体外研究，但2002年的研究表明，粗神经孢子虫CYT-19蛋白在线粒体I族内含子的折叠中起作用。进一步的研究表明，CYT-19也可以与II组内含子有效相互作用，表明它具有一般的RNA伴侣活性，相关的酵母蛋白Mss116p被证明具有类似的功能。本项目的目标是使用定义良好，易于处理的CYT-19和I组RNA系统来剖析DExD/H-box蛋白的RNA伴侣活性机制。除了增加对基本细胞过程的了解外，这项工作还对疾病有影响，因为DExD/H-box蛋白是包括HCV在内的病毒复制所必需的，而人类DExD/H-box蛋白的过表达与结肠癌和前列腺癌有关。在当前资助期间的进展导致了RNA伴侣活性的一般模型，这是本提案的中心。首先，CYT-19可以不加区分地破坏RNA结构，揭示非同源I族内含子的天然状态和长寿命错误折叠的构象，其效率取决于RNA种类的相对稳定性，而不取决于任何特定的结构特征。然而，它的活动并非完全不分青红皂白；它通过形成额外的“栓系”相互作用优先作用于结构化RNA，并且只有当P1没有与RNA的其他部分“停靠”到三级接触时，它才能解开I组内含子（P1）的短螺旋。这些结果表明，CYT-19可能会破坏无法正确包装的优先错误折叠rna。本研究的目标是进一步深入探究DExD/H-box蛋白对RNA伴侣活性的物理基础，探索CYT-19及其生理底物的特异性，并检验关于叔接触系结和抑制的机制和影响的假设。具体目的是：1)利用寡核苷酸置换法和定向羟基自由基足迹法探测I族内含子上RNA伴侣活性的途径；2)确定CYT-19是否以非同源RNA缺乏的特异性作用于其同源I族内含子；3)进一步探讨三级接触形成的抑制作用，包括通过确定对不同结构元件和不同I族内含子是否存在抑制作用来验证抑制作用是伴侣活性的一般特征的假设；4)验证拴系相互作用是由CYT-19和相关的DExD/H-box蛋白的高度碱性“尾巴”形成的假设，并且这种相互作用在局部解绕期间可以保持完整。这些结果有望指导DExD/H-box蛋白在RNA代谢的各个方面的模型。

DESCRIPTION (provided by applicant): DExD/H-box proteins are required for virtually every process carried out by structured RNAs, from pre-mRNA splicing and translation to intracellular trafficking of proteins and RNAs. These proteins are thought to use energy from ATP to facilitate RNA conformational changes and folding transitions, but relatively little is known on a molecular level about how they manipulate RNA structure. While many of the RNA and RNA-protein targets of DExD/H-box proteins are large, complex, and difficult to study in vitro, it was shown in 2002 that the Neurospora crassa CYT-19 protein functions in folding of several mitochondrial group I introns. Further work indicated that CYT-19 can also interact productively with group II introns, indicating that it possesses general RNA chaperone activity, and the related yeast protein Mss116p was shown to function similarly. The goal of this project has been to use the well-defined, tractable system of CYT-19 and group I RNAs to dissect the mechanisms of RNA chaperone activity by DExD/H-box proteins. In addition to increasing knowledge of essential cellular processes, this work has implications for diseases, as DExD/H-box proteins are required for replication of viruses including HCV, and overexpression of human DExD/H-box proteins is linked to colon and prostate cancer. Progress during the current funding period led to a general model for RNA chaperone activity, around which this proposal is centered. First, CYT-19 can disrupt RNA structure indiscriminately, unfolding both the native state and a long-lived misfolded conformer of a non-cognate group I intron with efficiencies that depend on the relative stabilities of the RNA species but not on any specific structural features. However, its activity is not fully indiscriminate; it acts preferentially on structured RNAs by forming an additional 'tethering' interaction, and it unwinds a short helix of a group I intron (P1) only when P1 does not 'dock' into tertiary contacts with the rest of the RNA. These results suggest that CYT-19 may disrupt preferentially misfolded RNAs that cannot pack correctly. The goals of the current proposal are to delve further into the physical basis of RNA chaperone activity by DExD/H-box proteins, to probe for specificity using CYT-19 and its physiological substrates, and to test hypotheses on the mechanisms and implications of tethering and inhibition by tertiary contacts. Specific Aims are: 1) to use an oligonucleotide displacement assay and directed hydroxyl radical footprinting to probe pathways of RNA chaperone activity on group I introns; 2) to determine whether CYT-19 acts on its cognate group I introns with specificity that is absent with a non-cognate RNA; 3) to probe further the inhibition by tertiary contact formation, including testing the hypothesis that the inhibition is a general feature of chaperone activity by determining whether it is present for different structural elements and within a different group I intron; and 4) to test the hypotheses that the tethering interaction is formed by a highly basic 'tail' of CYT-19 and related DExD/H-box proteins, and that this interaction can remain intact during local unwinding. Results are expected to guide models for DExD/H-box proteins in all aspects of RNA metabolism. PUBLIC HEALTH RELEVANCE: The goal of this project is to understand how RNA chaperone proteins assist RNAs as they fold to specific structures and exchange between structures. These proteins are required for viral replication and are linked to human cancer, so understanding how they function is important for understanding and ultimately treating human disease.